Multicolor Fluorescence Reader with Dual Excitation Channels

ABSTRACT

Provided is a fluorescence reader that uses two excitation channels and can read up to seven different fluorescent dyes in a single run. Each excitation channel has one light source and one single excitation filter and one dichroic mirror. One excitation channel is capable of exciting multiple fluorescent dyes and can be used to distinguish multiple dyes in combination with multiple emission filters. The excitation channels are driven by a motor that can automatically switch the two excitation channels for taking images of up to seven different fluorescent dyes. An algorithm to calibrate the crosstalk between different fluorescent dyes is also provided. Also provided is a method for analyzing digital PCR data using a ratio of two fluorescence emission readings.

FIELD OF THE INVENTION

This invention relates to the field of fluorescence microscopy,especially relates to a multicolor fluorescence reader with dualexcitation channel.

BACKGROUND OF THE INVENTION

Fluorescence microscopy is an essential tool in the field of lifesciences that uses fluorescence to generate an image of a sample, whichhas endogenous or exogenous fluorophores, chemical compounds that canre-emit light upon light excitation. Its principle involves illuminatinga sample with an excitation light of a specific wavelength bandwidth anddetecting the emission light of a longer wavelength that is emitted fromthe sample irradiated by the excitation light. At the core of thistechnology is to use a set of filters to direct the excitation light tothe sample and selectively let the emission light reach the detectorwhile blocking the excitation light from doing the same. The set offilters include an excitation filter, a dichroic mirror, and an emissionfilter. During the fluorescence imaging, the source light passingthrough an excitation filter shines onto a dichroic mirror whichreflects the excitation light to the sample. Upon illumination, thesample emits an emission light that passes through the dichroic mirrorand an emission filter and is received by the detector. The excitationlight cannot pass through the dichroic mirror or the emission filter,and is therefore blocked from reaching the detector. For a particularfluorescent dye, it usually has a set of an excitation filter, anemission filter and a dichroic mirror that optimally suits the physicalproperty of the dye (e.g. the excitation and the emission spectra of thedye) and can generate images with the highest signal to noise ratio.

Multicolor fluorescence imaging has many applications, for example,visualization of subcellular structures and multiplexed digital PCRdetection. The multicolor imaging techniques, when implementedsuccessfully, can provide rich information about the subject ofinterest. However, there are challenges and limitations that hinders thewide application of this powerful technique. For multiple fluorescentdyes used in the multicolor imaging, multiple sets of filters areusually required to be installed in a microscope, leading to complicatedand expensive machines that can be afforded only to a few luckylaboratories or institutes. It is also very difficult to find a group ofdyes with manageable crosstalk among each other and to correct thecrosstalk among multiple dyes. As a result, four-color fluorescencemicroscope is the most commonly found product on the market. It is hardto find a multicolor fluorescence microscope that can support more thanfive colors. There is a need to develop a simple and inexpensivefluorescence microscope that can support simultaneous imaging of morethan five colors. The present invention satisfies this need and providesother benefits as well.

SUMMARY OF THE INVENTION

Provided is a multicolor fluorescence reader that uses two excitationchannels and can read up to seven different fluorescent dyes in a singlerun. Each excitation channel has one light source and one singleexcitation filter and one dichroic mirror. One excitation channel iscapable of exciting multiple fluorescent dyes and can be used to readmultiple dyes in combination with multiple emission filters. Theexcitation channels are driven by a motor that can automatically switchthe two excitation channels for taking images of up to seven differentfluorescent dyes. An algorithm to calibrate the crosstalk betweendifferent fluorescent dyes is also provided.

In one embodiment of the invention, there provides a multicolorfluorescence reader, comprising an emission filter wheel and twoexcitation channels that can be automatically moved, wherein eachexcitation channel has a light source, an excitation filter, and adichroic mirror, and wherein the multicolor fluorescence reader can readup to seven different fluorophores in a single run.

In some embodiments, the excitation channel is moved by a stepper motorvia connecting gears.

In some embodiments, the emission filter wheel of the multicolorfluorescence reader has three, four, five, six or more emission filters.The emission filter wheel is driven by a stepper motor to switchdifferent emission filters while taking images for different fluorescentdyes.

In some embodiments, the light source used in the multicolorfluorescence reader is selected from light-emitting diode (LED) lamps,xenon arc lamps and mercury-vapor lamps.

In some embodiments, the light source in a first excitation channel is ahigh-power LED lamp with a peak wavelength of <500 nm and the lightsource in a second excitation channel is a high-power LED lamp with apeak wavelength of >580 nm.

In some embodiments, the first excitation channel can be used to excitefive different fluorophores and the second excitation channel can beused to excite two different fluorophores.

In some embodiments, the multicolor fluorescence reader is equipped withan emission filter wheel with six emission filters.

In some embodiments, the multicolor fluorescence reader has six emissionfilters that can be used to read seven fluorophores, for example, FAM,VIC, ABY, JUN, Cy5, Cy5.5 and ATTO.

In some embodiments, the multicolor fluorescence reader comprises a PCRchip holder that can hold multiple PCR chips.

In some embodiments, the multicolor fluorescence reader comprises aslide holder that can hold multiple glass slides.

In some embodiments, the crosstalk between two fluorophores can becorrected by a calibration constant which is empirically determined. Thecrosstalk between fluorophore A and B is corrected as follows:

F _(A) ′=F _(A) −K _(B→A) *F _(B)

F _(B) ′=F _(B) −K _(A→B) *F _(A)

Wherein F_(A) and F_(B) are raw fluorescence intensity of A and B,respectively; F_(A)′ and F_(B)′ are corrected fluorescence intensity ofA and B, respectively; K_(B→A) is the calibration constant forcorrecting bleed-through from fluorescence channel B to A; and K_(A→B)is the calibration constant for correcting bleed-through fromfluorescence channel A to B.

In some embodiments of the invention, there provides a method forcorrecting bleed-through from n different fluorescence channels intofluorescence channel A in multicolor fluorescence recordings, whereincorrected fluorescence intensity of fluorescence channel A is calculatedas following:

F _(A) ′=F _(A)−Σ_(i=1) ^(n)(K _(i) *F _(i))

wherein F_(A) is raw fluorescence intensity of fluorescence channel A;F_(A)′ is corrected fluorescence intensity of fluorescence channel A;K_(i) is calibration constant for correcting bleed-through from i^(th)fluorescence channel to fluorescence channel A; F_(i) is rawfluorescence intensity of i^(th) fluorescence channel, and n is thenumber of different fluorescence channels that have bleed-through intofluorescence channel A, i is an integer from 1, 2, . . . to n, whereinthe calibration constant K_(i) for correcting bleed-through from afluorescence channel to fluorescence channel A is empiricallydetermined.

In some embodiments, it provides a the method for determining acalibration constant (K_(i)) for correcting bleed-through from i^(th)fluorescence channel to fluorescence channel A comprises the steps of:a, obtaining a plurality of multicolor fluorescence recordings with thefluorescence intensity of fluorescence channel A (F_(A)) and thefluorescence intensity of i^(th) fluorescence channel (F_(i)); b,displaying, on a scatter plot, adjusted fluorescence intensity offluorescence channel A (F_(Adj)) and the fluorescence intensity ofi^(th) fluorescence channel (F_(i)), wherein F_(Adj)=F_(A)−N_(i)*F_(i),wherein N_(i) is a value that can be changed; c, varying N_(i), inresponse to user input, and observing the change of the shape of thescatter plot of F_(Adj) and F_(i); and d, selecting the value of N_(i)as the calibration constant (K_(i)) for correcting bleed-through fromi^(th) fluorescence channel to fluorescence channel A when the shape ofthe scatter plot is most close to flat status.

In one embodiment of the invention, it provides a method of analyzingdigital PCR data, comprising: a, measuring a fluorescent emission ineach micro-well of a dPCR chip before the start of a digital PCR(F_(s)); b, measuring a fluorescent emission in each micro-well of thedPCR chip at the end of the digital PCR (F_(e)); c, determining anemission ratio T=F_(e)/F_(s) for each micro-well; d, using a thresholdvalue for the emission ratio T to identify micro-wells with positive PCRamplifications; and e, calculating a percentage of positive PCRamplification as a result of the digital PCR.

In some embodiments, F_(s) and F_(e) are measurements of the samereporter fluorescent dye.

In some embodiments, F_(s) is a measurement of a passive fluorescent dyeand F_(e) is a measurement of a reporter fluorescent dye.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a side section view of a fluorescence microscope embodiment ofthe invention showing the major components, including dual excitationchannel having two LED light sources (1), two emission filters (2) andtwo dichroic mirrors (3), an emission filter wheel (4), a camera (5),PCR chips (6), a PCR chip holder (7) and a controlling motor for theexcitation channels (8).

FIG. 2 is an inside view of the fluorescence microscope showing thepositioning of two LED light sources (1) and two excitation filters (2),two dichroic mirrors (3), a camera (5), PCR chips (6), a PCR chip holder(7), a controlling motor for the excitation channels (8), and a holdingtrack for the excitation channels (9).

FIG. 3 is a top view of the machine showing the first excitation channel(10), the second excitation channel (11), a stepper motor for moving theexcitation channel (12), gears turned by the stepper motor to move theexcitation channel (13), and a holding track along which the twoexcitation channels can be moved (9).

FIG. 4 is a top view of an emission filter wheel (4) that has sixemission filters (14) driven by a stepper motor (15) using a connectingpinion (16).

FIG. 5 shows a linearity analysis of the concentration of a fluorescencedye (VIC) and the measured fluorescence intensity. The x-axis and y-axisrepresent the concentration of VIC-labeled oligo nucleotide and thefluorescence intensity, respectively.

FIG. 6 shows a linearity analysis of the concentration of a fluorescencedye (FAM) and the measured fluorescence intensity. The x-axis and y-axisrepresent the concentration of FAM-labeled oligo nucleotide and thefluorescence intensity, respectively.

FIG. 7 shows a test result of how fluorescent dye FAM affectsmeasurement readings of other fluorescent dyes including VIC, ABY, ROXand ATTO.

FIG. 8 shows a test result of how fluorescent dye VIC affectsmeasurement readings of other fluorescent dyes including FAM, ABY, ROXand ATTO.

FIG. 9 shows a linearity analysis of a six-plex digital PCR experiment.Six different target sequences in a sample were detected in the digitalPCR assay by six different fluorescent probes labeled by FAM, VIC, ATTO,ABY, ALEXA, or Cy5.5. The x-axis represents the positive count of targetsequences determined by the digital PCR assay. The y-axis represents theamount of input target sequence added by serial dilution.

DETAILED DESCRIPTION

Abbreviations: DNA—deoxyribonucleic acid; RNA—Ribonucleic acid; andPCR—polymerase chain reaction.

Definitions: Unless otherwise defined, all technical and scientificterms used herein have the same meaning as commonly understood by one ofthe ordinary skill in the art to which this invention belongs.

The term “a” and “an” and “the” as used to describe the invention,should be construed to cover both the singular and the plural, unlessexplicitly indicated otherwise or clearly contradicted by context.Similarly, plural terms as used to describe the invention, should alsobe construed to cover both the plural and the singular, unless indicatedotherwise or clearly contradicted by context.

The term “excitation channel”, as used herein, refers to a set ofoptical components in a fluorescence reader that are used to send anexcitation light of specific bandwidth to a sample to be tested. Anexcitation channel usually includes a light source, an excitation filterand a dichroic mirror. There may be multiple light sources, excitationfilters and dichroic mirrors in one excitation channel. The excitationchannel of the invention has a single light source, a single excitationfilter and a single dichroic mirror.

Majority of the optical systems commonly used for fluorescent microscopyrequires expensive filter sets to allow simultaneous detection ofmultiple fluorescent dyes. Each filter set has an excitation filter, anemission filter and a dichroic mirror optimized for the excitation andemission spectra of a single fluorophore. Multicolor fluorescent imagingis limited by the number of the filter sets that can be accommodated bythe microscope. High number of filter sets also significantly increasesthe cost of the microscope. Some system uses laser of differentwavelengths to excite different fluorophores. The number of colors thatcan be measured is limited to the availability of lasers and thecompatibility of different fluorescent dyes. Increase of the number oflasers increases the complexity of the microscope and also contributesto the increase of the instrument cost. This is why the most commonmulticolor fluorescent imaging is less than four colors. Thefluorescence microscope that can image more than 5 colors is very rareto find.

In one embodiment of the invention, it provides a fluorescence readerusing two excitation channels in combination of emission filters to readup to seven fluorophores. Each excitation channel includes a lightsource, an excitation filter and a dichroic filter. One excitationchannel produces an excitation light of a shorter wavelength (e.g. <500nm) that can be used to excite five categories of commonly usedfluorescent dyes such as FAM, VIC, ABY, ATTO and JUN. Another excitationchannel produces an excitation light of a longer wavelength (e.g. >600nm) that can be used to excite fluorescent dyes with longer excitationwavelength such as Cy5.5 and Cy5. The fluorescent dyes excited by thesame excitation channel is distinguished by different emission filters.This setup can provide excitation lights of both shorter and longerwavelengths without extensive use of excitation filters. It provides asimple and easy to operate alternatively to the currently availablemodels. It can easily measure up to seven different fluorescent dyeswith only a portion of the cost.

In one embodiment of the invention, it provides a multicolorfluorescence reader that can read up to seven different fluorophores ina single run, comprising an emission filter wheel and two excitationchannels that can be automatically moved, wherein each excitationchannel has a light source, an excitation filter, and a dichroic mirror.As shown in FIGS. 1-3, the two excitation channels (10&11) are placedside by side underneath the camera (5). Samples are placed below theexcitation channels and the emission filter wheel (4) is positionedbetween the camera (5) and the excitation channels (10&11). Samples usedin the fluorescence reader can be, for example, digital PCR chips,microarrays slides, cell staining slides, etc. In some embodiments, thefluorescence reader is equipped with a digital PCR chip holder, whichcould hold, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more PCRchips. In some embodiments, the fluorescence reader is equipped with aglass slide holder, which could hold, for example, 1, 2, 3 or 4 glassslides. Each excitation channel packed as one unit has a single lightsource (1), a single excitation filter (2), and a dichroic mirror (3),which can be moved along a holding track (9) by a controlling motor (8).The controlling motor (8) comprises a stepper motor (12) and connectinggears (13) driven by the stepper motor (12). The controlling motor canbe programmed to move one of the two excitation channels to be directlyunderneath the camera for picture taking. During the imaging process,the light source sends out a light beam that is filtered by theexcitation filter. The pass-through light, the excitation light, isreflected by the dichroic mirror to shine onto the sample. Uponillumination by the excitation light, the sample emits an emission lightof longer wavelength that can pass through the dichroic mirror. Theemission light is further filtered by a selected emission filter and thepass-through emission light will be detected by the camera. Both thedichroic mirror and the emission filter are used to block the excitationlight and prevent it from reaching the camera.

The first excitation channel can generate an excitation light withshorter wavelength and the second excitation channel can generate anexcitation light with longer wavelength so that a wide spectrum offluorescent dyes can be excited using both excitation channels. Forexample, the first excitation channel can use a blue LED lamp with apeak wavelength shorter than 500 nm (e.g. 460 nm, 465 nm, 470 nm, 475nm, 480 nm, or 490 nm) to excite commonly used fluorescent dyes such asFAM, VIC, ABY, ATTO and JUN. An excitation filter with a band pass of 20nm, 30 nm, 40 nm, 50 nm or 60 nm can be used to filter the LEDexcitation light. A dichroic mirror selected should be able to reflectthe excitation light with a wavelength of, for example, 430-490 nm,440-500 nm, 450-520 nm, or 420 nm-500 nm, and pass through emissionlights of the fluorescent dyes used. The lights emitted from thefluorescent dyes are further filtered by respective emission filters onthe emission filter wheel.

The second excitation channel can use a red LED lamp with a peakwavelength longer than 580 nm (e.g. 580 nm, 610 nm, 615 nm, 617 nm, or620 nm) to excite fluorescent dyes that can be excited by longwavelength lights such as Cy5.5 and Cy 5. An excitation filter of anappropriate band pass is used to filter the LED excitation light. Theexcitation filter is chosen to pass through an excitation light that canefficiently excite the fluorescent dyes of choice and have no overlapwith the bandwidth of the emission lights of the fluorescent dyes. Thewavelength band pass of the excitation filter can be, for example,575-620 nm, 590-640 nm or 585-635 nm. The dichroic mirror selected forthe second channel should be able to deflect long wavelength excitationlight having a deflection range of, for example, 500-630 nm, 400-620 nm,or 450-625 nm. The pass-through wavelength range of the dichroic mirroris selected depending on the emission wavelengths of the fluorescentdyes.

FIG. 4. shows a top view of the emission filter wheel (4) that comprisessix emission filters (14), which can be rotated by a motor to select thecorrect emission filter for a particular fluorescent dye. The motor forrotating the emission filter wheel comprises a stepper motor (15) andconnecting pinion (16). The stepper motor moves the connection pinion,which in turn moves the emission filter wheel. The emission filter wheelmay contain 4, 5, 6, 7, 8 or more emission filters. Since multiplefluorescent dyes are excited by the same excitation channel, they can bedistinguished by narrow-banded emission filters.

An emission filter for a fluorescent dye is usually set around the peakwavelength of an emission light. It can also be set at a wavelengthrange apart from the peak wavelength in order to separate twofluorescent dyes with close emission spectra. The bandwidth of anemission filter can be, for example, 10 nm, 15 nm, 20 nm, 25 nm, 35 nmor 40 nm. For fluorescent dyes having close emission spectra, thebandwidths of emission filters need to be set to a narrower range (e.g.8 nm or 10 nm) so that the emission lights for the two fluorescent dyescan be distinguished. If two neighboring fluorescent dyes have widespread emission spectra, the bandwidth of emission filter chosen canhave a wider range, for example, 35 nm or 40 nm.

Sometimes, two fluorescent dyes can share the same emission filterbecause they are excited at different wavelengths. For example, ATTO andCy5 can be excited by the first and second excitation channel,respectively. The ATTO and Cy5 have emission lights with peakwavelengths at 658 nm and 676 nm, respectively, which can share anemission filter that covers the peak wavelength for both fluorescentdyes. This setting allows using six emission filters and two excitationchannels to separately measure seven different dyes. The sevenfluorescent dyes are measured sequentially, and the imaging data can becombined in the analysis afterwards. The crosstalk between multiplefluorescent dyes can be corrected by a calibration constant, which isdetermined empirically.

FIGS. 5 and 6 show linearity analysis of two fluorescent dyes, FAM andVIC. It was tested to see if the measured fluorescence intensity and theconcentration of fluorescent dye has a good linear relationship.Different concentrations of oligonucleotides labeled with a singlefluorescent dye were measured in the fluorescence reader, and theconcentration of the dye is plotted against the measured fluorescenceintensity. The results show that the concentrations of both fluorescentdyes have very good linear relationship with the fluorescencemeasurement. The coefficient of determination (R²) of both dyes arehigher than 0.99, indicating a very good linear relationship. The sameexperiments were also done for other five fluorescent dyes. All of themhave good linearity with R² values higher than 0.99. These resultsindicate that the concentration of the fluorescent dye and the measuredfluorescence has a very good linear relationship.

When multiple fluorescent dyes are measured, it is important to testwhether the signal of a fluorescent dye is bled through into the signalof another and make correction of the crosstalk. FIG. 7 shows the resultof a method for testing if the signal of fluorescent dye is bled throughinto other fluorescent channels. The method tests if the increase ofsignals of a single fluorescent dye can lead to increase of signals inanother fluorescent channel. If the answer is yes, it indicates that thefluorescent dye of the test may bleed through into another fluorescentdye, and calibration of the signal bleed-through needs to be done. Ifthe answer is no, it indicates that the increase of the fluorescent dyeof the test does not have any effect on the other fluorescent dye, andit is likely that the tested fluorescent dye does not bleed through intothe channel of other dye.

A digital PCR experiments was performed in a digital PCR chip using onlyone single fluorescent labeling of FAM. The fluorescent reader read thefluorescence intensity for FAM, VIC, ABY, ROX and ATTO in 20,000 wellsof the digital PCR chip. There is no VIC, ABY, ROX or ATTO in the wellsof the digital PCR chip. If there is no bleed-through from FAM to otherfluorescence channel, the readings in VIC, ABY, ROX and ATTO channelshould show only background noise. On the other hand, if there isbleed-through from FAM to another fluorescence channel, the affectedchannel will have higher readings with increase of FAM signals. Themeasured FAM intensity of all the wells were sorted from lowest (left)to highest (right). The corresponding intensity readings of the alignedwells for all the fluorescent dyes were plotted in FIG. 7. When themeasured FAM intensity was increased, the fluorescent intensity readingsin the corresponding wells for ROX and ATTO did not have significantchange while the fluorescent intensity readings for VIC and ABY weresignificantly increased with the increase of FAM fluorescence readings.These results indicate that FAM signals do not bleed into thefluorescence channel of ROX and ATTO, and FAM signals do bleed into thefluorescence channel of VIC and ABY. The same experiments were performedfor other fluorescent dyes as well. FIG. 8 shows another example oftesting if VIC fluorescence signals bleed into other fluorescencechannels. The results indicate that VIC fluorescence signals may havesmall bleed-through into the fluorescence channel of ATTO, but not intofluorescence channels of other dyes.

The crosstalk calibration among multiple fluorescent dyes should be donein a stepwise manner. The first step is to determine if two fluorescentdyes have crosstalk. The second step is to first calibrate the crosstalkbetween two fluorescent dyes that have the closest emission spectra, andthen calibrate with the dye with the next closest emission spectra,until all the crosstalk is corrected. Usually, the raw fluorescenceintensity readings are corrected for factors such as light evenness,shape of the well, physical artifacts before the crosstalk calibration.For example, the raw fluorescence intensity readings can be corrected bydividing the raw value with a reference value, if available. For digitalPCR experiment, a reference dye, ROX, are evenly added to all the wellsin a digital chip. The ROX reading can then be used as the referencevalue.

The key for calibrating crosstalk between two fluorescent dyes is tofind a calibration constant for bleed-through from one dye to another.Once the calibration constant is determined, the crosstalk betweenfluorophore A and B is corrected as follows:

F _(A) ′=F _(A) −K _(B→A) *F _(B)  (a)

F _(B) ′=F _(B) −K _(A→B) *F _(A)  (b)

wherein F_(A) and F_(B) are raw fluorescence intensity of A and B,respectively; F_(A)′ and F_(B)′ are corrected fluorescence intensity ofA and B, respectively; K_(B→A) is the calibration constant forcorrecting bleed-through from fluorescence channel B to A; and K_(A→B)is the calibration constant for correcting bleed-through fromfluorescence channel A to B. The calibration constant is determinedempirically. For example, if a fluorescence channel A is bled into thefluorescence channel of B, the plot of readings of fluorescence dye Avs. readings of fluorescent dye B will have a linear relationship with aslop bigger than 0. The bigger the slope, the more bleed-through fromthe fluorescence channel A into B. To correct for the bleed-through of Ainto B, different calibration constants from 0 to 1 can be tested in theequation (b) to obtain corrected F_(B)′. Plot the F_(A) vs. thecorrected F_(B)′ to obtain a slope of the linear relationship. Thecorrect final calibration constant (K_(A→B)) should make the slope ofthe above plot be closest to 0. The intensity value corrected for A to Bbleed-through will be F_(B)′=F_(B)−K_(A→B)*F_(A). Sequentially correctthe bleed-through from fluorescence channel of other fluorescent dyes(e.g. C, D), and the corrected final value for fluorescent dye B will bedetermined, that is,

F _(B) ′=F _(B) −K _(A→B) *F _(A) −K _(C→B) *F _(C) −K _(D→B) *F _(D)

The same calibration procedure should be applied to every fluorescentdye to obtain the corrected value for each dye.

In some embodiments, there provides a method for correctingbleed-through from n different fluorescence channels into fluorescencechannel A in multicolor fluorescence recordings, wherein correctedfluorescence intensity of fluorescence channel A is calculated asfollowing:

F _(A) ′=F _(A)−Σ_(i=1) ^(n)(K _(i) *F _(i))

wherein F_(A) is raw fluorescence intensity of fluorescence channel A;F_(A)′ is corrected fluorescence intensity of fluorescence channel A;K_(i) is calibration constant for correcting bleed-through from i^(th)fluorescence channel to fluorescence channel A; F_(i) is rawfluorescence intensity of i^(th) fluorescence channel, and n is thenumber of different fluorescence channels that have bleed-through intofluorescence channel A, wherein the calibration constant for correctingbleed-through from a fluorescence channel to fluorescence channel A isempirically determined.

In some embodiments, a method is provided for determining a calibrationconstant (K_(i)) for correcting bleed-through from i^(th) fluorescencechannel to fluorescence channel A. The method comprises the steps of: a,obtaining a plurality of multicolor fluorescence recordings with thefluorescence intensity of fluorescence channel A (F_(A)) and thefluorescence intensity of i^(th) fluorescence channel (F_(i)); b,displaying, on a scatter plot, adjusted fluorescence intensity offluorescence channel A (F_(Adj)) and the fluorescence intensity ofi^(th) fluorescence channel (F_(i)) of the plurality of multicolorfluorescence recordings, wherein F_(Adj)=F_(A)−N_(i)*F_(i), whereinN_(i) is a variable that can be changed; c, varying N_(i), in responseto user input, and observing the change of the shape of the scatter plotof F_(Adj) vs. F_(i); and d, selecting the value of N_(i) as thecalibration constant (K_(i)) for correcting bleed-through from i^(th)fluorescence channel to fluorescence channel A when the shape of thescatter plot is most close to flat status.

The method can be implemented either manually or by a computer-basedprogram. For manual implementation, one can change N_(i) from 0 to apositive value in a stepwise manner. For each selected N_(i), make ascatter plot of F_(adj) vs. F_(i) of a plurality of multicolorfluorescence recordings. If there is bleed-through from fluorescencechannel i into fluorescence channel A, the intensity in fluorescencechannel A is increased with the increase of intensity in fluorescencechannel i. In a scatter plot of the two fluorescence intensities, theshape of scattered dots is slanted upwards, indicative of positivecorrelation between the two fluorescence intensities. Find an N_(i) foradjusting the F_(A) such that the correlation between the twofluorescence intensities (F_(adj) and F_(i)) is at the minimum level. Ina scatter plot of the adjusted fluorescence intensity F_(adj) vs. F_(i),the shape of scattered dots should be at minimum slanted upwards,ideally, be at a flat status. When such an N_(i) is found, it isselected as the calibration constant K_(i) for correcting thebleed-through from the fluorescence channel i to the fluorescencechannel A. The method can be repeated until all the calibrationconstants are determined for any two fluorescence channels withcrosstalk.

The method can also be implemented by a computer program. The computerprogram provides a graphic user interface for user to choose any twofluorescence channels (F_(A) and F_(i)) to perform crosstalkcalibration. It provides a functional bar for changing the value ofN_(i) along with a display of the corresponding scatter plot of the twoselected fluorescence channels (F_(adj) and F_(i)). When a user changesthe value of N_(i), the change of fluorescence intensities(F_(Adj)=F_(A)−N_(i)*F_(i)) is directly reflected in the companyingscatter plot, which enables the user to select an appropriate N_(i) asthe calibration constant based on the change of the shape of thescattered dots in the scatter plot. The program allows a user to adjustN_(i) while visually inspecting the change of adjusted data so that theuser can easily find an appropriate calibration constant for twochannels with crosstalk.

A chip-based digital PCR is carried out by partitioning a sample into alarge number of micro-wells of a digital PCR chip to perform a largenumber of PCR microreactions in parallel. The positive amplification isdetermined at the end of the digital PCR by detecting fluorescentchemicals generated during PCR amplifications in the micro-wells. Thisis called an end-point digital PCR where only one measurement is done atthe end of the reaction. The end-point fluorescent emission readings arecompared to a threshold and those with end-point fluorescent emissionreadings higher than the threshold are determined to have positiveamplifications. Since this method uses only one measurement to determinepositive/negative outcomes, variation factors among differentmicro-wells, including volume variations, differences in position andshape, and fluorescent dye concentration variations, can contribute tovariations in the final result, leading to high incidences of falsepositives and false negatives. To minimize and compensate for theeffects of these variation factors, a method is provided to measurefluorescent emissions before and after a digital PCR and use the ratioof the two fluorescent emission readings as the determinant fordetecting positive/negative amplifications.

In one embodiment of the invention, there provides a method of analyzingdigital PCR data, comprising the steps of: a, measuring a fluorescentemission in each micro-well of a dPCR chip before the start of a digitalPCR (F_(s)); b, measuring a fluorescent emission in each micro-well ofthe dPCR chip at the end of the digital PCR (F_(e)); c, determining anemission ratio T=F_(e)/F_(s) for each micro-well; d, using a thresholdvalue for the emission ratio T to identify micro-wells with positive PCRamplifications; and e, calculating a percentage of positive PCRamplification as a result of the digital PCR.

In a digital PCR system, more than one reporter fluorescent dyes alongwith a passive fluorescent dye are usually employed to detect targetgeneration. Emissions from reporter fluorescent dyes are directlycorrelated with PCR generation of nucleic acids while emission from thepassive fluorescent dye is not related with PCR generation of nucleicacids. Measurement of both reporter fluorescent dyes and passivefluorescent dye are made before and after a digital PCR. F_(s) is afluorescent emission reading made before the start of the digital PCR.It can be an emission reading of a reporter fluorescent dye or a passivefluorescent dye. F_(e) is a fluorescent emission reading made after theend of the digital PCR. It is an emission reading of a reporterfluorescent dye. Preferably, F_(e) and F_(s) are crosstalk calibratedemission readings. The ratio F_(e)/F_(s) is used as a determinant toidentify positive and negative amplifications by comparing to athreshold. The threshold ratio is selected as a value that is higherthan the ratio of background level. The method for selecting a thresholdin digital PCR analysis is well known to the one with ordinary skill inthe art. For example, US20150269756A1 discloses a method of selecting athreshold for digital PCR analysis using a scatter plot or a histogramof fluorescent emission readings. When the ratio F_(e)/F_(s) of amicro-well is higher than a threshold, the micro-well is considered tohave a positive PCR amplification, that is, it contains a target nucleicacid sequence. When the ratio F_(e)/F_(s) of a micro-well is smallerthan a threshold, the micro-well is considered to have a negative PCRamplification, that is, it does not contain a target nucleic acidsequence. In some embodiments, F_(s) and F_(e) are measurements of thesame reporter fluorescent dye. In other embodiments, F_(s) is ameasurement of a passive fluorescent dye and F_(e) is a measurement of areporter fluorescent dye. After micro-wells with positive amplificationsare identified, the percentage of positive micro-wells can be calculatedto be the result of the digital PCR.

FIG. 9 shows a linearity analysis of a six-plex digital PCR experimentusing six different fluorescent probes to detect six different targetsequences in the same sample. All the six fluorescent probes (FAM, VIC,ATTO, Cy5.5, Alexa and Aby) showed good linearity with R² value higherthan 0.99. The fluorescent emissions were read by the multicolorfluorescence reader of the invention and fluorescence data werecalibrated for crosstalk using the methods described herein. Thefluorescence reader successfully read emissions from seven fluorescentdyes in a sample, including six reporter fluorescent dyes and a passivefluorescent dye.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables,appendices, patents, patent applications and publications, referred toabove, are hereby incorporated by reference.

What is claimed is:
 1. A multicolor fluorescence reader, comprising anemission filter wheel and two excitation channels that can beautomatically moved, wherein each excitation channel has a light source,an excitation filter, and a dichroic mirror, wherein the multicolorfluorescence reader can read up to seven different fluorophores in asingle run.
 2. The multicolor fluorescence reader of claim 1, whereinthe excitation channel is moved by a stepper motor.
 3. The multicolorfluorescence reader of claim 1, wherein the emission filter wheel hasthree, four, five, six or more emission filters.
 4. The multicolorfluorescence reader of claim 1, wherein the light source is selectedfrom light-emitting diode (LED) lamps, xenon arc lamps and mercury-vaporlamps.
 5. The multicolor fluorescence reader of claim 4, wherein thelight source in a first excitation channel is a high-power LED lamp witha peak wavelength of <500 nm and the light source in a second excitationchannel is a high-power LED lamp with a peak wavelength of >580 nm. 6.The multicolor fluorescence reader of claim 5, wherein the firstexcitation channel can be used to excite five different fluorophores andthe second excitation channel can be used to excite two differentfluorophores.
 7. The multicolor fluorescence reader of claim 6, whereinthe emission filter wheel has six emission filters.
 8. The multicolorfluorescence reader of claim 6, wherein the multicolor fluorescencereader can measure emissions from seven fluorophores of FAM, VIC, ABY,JUN, Cy5, Cy5.5 and ATTO.
 9. The multicolor fluorescence reader of claim1 further comprises a PCR chip holder that can hold multiple PCR chips.10. The multicolor fluorescence reader of claim 1 further comprises aslide holder that can hold multiple glass slides.
 11. A method forcorrecting bleed-through from n different fluorescence channels intofluorescence channel A in multicolor fluorescence recordings, whereincorrected fluorescence intensity of fluorescence channel A is calculatedas following:F _(A) ′=F _(A)−Σ_(i=1) ^(n)(K _(i) *F _(i)) wherein F_(A) is rawfluorescence intensity of fluorescence channel A; F_(A)′ is correctedfluorescence intensity of fluorescence channel A; K_(i) is calibrationconstant for correcting bleed-through from i^(th) fluorescence channelto fluorescence channel A; F_(i) is raw fluorescence intensity of i^(th)fluorescence channel, and n is the number of different fluorescencechannels that have bleed-through into fluorescence channel A, i is aninteger from 1, 2, . . . to n, wherein the calibration constant K_(i)for correcting bleed-through from a fluorescence channel to fluorescencechannel A is empirically determined.
 12. The method of claim 11, whereinthe method for determining a calibration constant (K_(i)) for correctingbleed-through from i^(th) fluorescence channel to fluorescence channel Acomprises the steps of: a, obtaining a plurality of multicolorfluorescence recordings with the fluorescence intensity of fluorescencechannel A (F_(A)) and the fluorescence intensity of i^(th) fluorescencechannel (F_(i)) b, displaying, on a scatter plot, adjusted fluorescenceintensity of fluorescence channel A (F_(Adj)) and the fluorescenceintensity of i^(th) fluorescence channel (F_(i)), whereinF_(Adj)=F_(A)−N_(i)*F_(i), wherein N_(i) is a value that can be changed;c, varying N_(i), in response to user input, and observing the change ofthe shape of the scatter plot of F_(Adj) and F_(i); and d, selecting thevalue of N_(i) as the calibration constant (K_(i)) for correctingbleed-through from i^(th) fluorescence channel to fluorescence channel Awhen the shape of the scatter plot is most close to flat status.
 13. Amethod of analyzing digital PCR data, comprising a, measuring afluorescent emission in each micro-well of a dPCR chip before the startof a digital PCR (F_(s)); b, measuring a fluorescent emission in eachmicro-well of the dPCR chip at the end of the digital PCR (F_(e)); c,determining an emission ratio T=F_(e)/F_(s) for each micro-well; d,using a threshold value for the emission ratio T to identify micro-wellswith positive PCR amplifications; and e, calculating a percentage ofpositive PCR amplification as a result of the digital PCR.
 14. Themethod of claim 13, wherein F_(s) and F_(e) are measurements of the samereporter fluorescent dye.
 15. The method of claim 13, wherein F_(s) is ameasurement of a passive fluorescent dye and F_(e) is a measurement of areporter fluorescent dye.